Anti-activated Notch1 antibody (ab8925)

Hello,
I have a question re your activated notch1 antibody.
You state that the predicted size of the protein is 80kDa in a western.
However, if you look at the cell signaliing anti-acivated Notch1 antibody
(http://www.cellsignal.com/products/2421.html),
you get a size of around 110 kDa.
This varies a bit,
depending on cell type used and post-translational modifications such as glykosilation etc.,
but with that antibody (which is used a lot), you never get 80kDa
(all in human cells, although mouse is the same).
How do you explain that?
Thanks,

ANSWER:

Thank you for your inquiry.

1.)Our antibody ab8925 is direct against the N-Terminal (aa 1755-1767) of the NOTCH 1 intracellular domain and molecular weights of 80 or 100 kd have been reported. The 80 kD is the expected MW for the transfected expressed intracellular domain of Notch1. As referenced in sequence for Notch 1 (SwissProt accession number P46531, see outbind://42/www.expasy.org), the intracellular domain is from amino acid 1754-2555 (802 aa, estimated ˜80-85 kD) and is the activated form of NOTCH1.

The 100 kD band shown in the figure corresponds to native Notch 1 intracellular domain as it is expressed in the samples. This is consistent with the molecular weight for this protein observed in un-transfected cells lysates which we have tested where the MW is in the range of 100-110 kD. It is possible different MW may be observed in particular samples, as modification of this domain is reported to occur after cleavage and the observed MW is still a matter for academic investigation.

2.) In the past, lysates have beenrun on a 4-20% gel under reducing conditions, transferred by standard methods and blocked for 1 hour 5%milk TTBS. Blotwas incubated with antibody ab8925 at a dilution of 1:200 overnight at 4 Cfollowed by a HRP conjuagetd secondary antibody. All conditions must be optimized by the user.

I hope this information is helpful and wish you good luck with your research.

I haven't heard anything from Fisher about the refund for the antibody (see below). Can you maybe touch base with them to see what is going on?

ANSWER:

Thank you for your reply.

I have talked to our accounts team and they have told me that we have generated a credit note for Fisher for ab8925. Therefore you would need to contact Fisher, with the PO number that you used with them to check on the status at their end.

If you have any issues when you contact Fisher, then please have them contact our accounts team at mailto:us.accounts@abcam.com and they will be able to help further.

Please let me know if there is anything else I can help you with.

Yes, for ab32144 we did staining on mouse lung sections using our previously described protocol.
Thank you for your help,age.

ANSWER:

Thank you for your reply.

I am sorry about the problems you have been having with these antibodies and as requested I have refunded the cost of ab32144. AS this antibody was ordered directly from by your institution, I am crediting the cost back to you and not Fisher as is the case with the other antibody I refunded.

The Credit Note number is ********.

Please pass this information onto your accounts department and if they have any questions about this please contact our accounts team at mailto:us.accounts@abcam.com.

If there is anything else I can help you with, please let me know.

We have tried to optimize the replacement antibody for Rb anti-activated Notch1 ab8925 and again we didn't see any staining. This is very weird and very frustrating as we had beautiful staining in the past with our original batch.
We also recently purchased a Rb mAb to CCR2 ab32144 . This antibody detects something nuclear in every single cell. I don't know what exactly it detects but not CCR2. This is very disappointing, so at this point we would like to get a refund for both antibodies.

ANSWER:

Thank you for your reply.

As the free of charge replacement we sent you did not prove to be successful, I am crediting back the cost of the original antibody (ab8925), from order number (this was placed onand the is

As this antibody was ordered through Fisher, the cost will be refunded to them and then they will have to credit that cost back to you. I have already emailed them to inform them about this and they should contact you.

As for ab32144, were you also using mouse lung sections and similar staining protocols as before?

We have recently purchased a new batch of the above antibody and are finding quite different results from the previous batch that we used.


Please find the attached file that shows blots using identical samples for overnight or 1hr incubations with the two batches of antibody.

You can see clearly that the NICD band appears to be upregulated in sample 1 when we used the old batch which is consistent with all previous findings from our group .This is not seen when using the new batch and in fact the nicd could even be interpreted as slightly down.

Unfortunately I do not have the lot number for the old batch but it was purchased on the ddmmyy.

i was wondering if you had any explanation of my findings

thank you

kath spence

ANSWER:

Thank you for contacting us.

I am sorry to hear you have been experiencing problems with one of our antibodies. The quality of our products is important to us and I would like to reassure you that we investigate all customer complaints.

Before attempting to find an explanation for the results you obtained with this whole antiserum of the rabbit polyclonal Notch1 antibody, I would like to ask some additional questions:

1a) What kind of samples are you investigating (species, endogenous/recombinant protein)? How have you prepared these samples and how did you store them?

1b) You mentioned that the blots show identical samples: Did you mean identical samples (on different blots) or are these even identical (i.e. stripped) blots? Also, what is the next MW marker above the 75 kDa band? I am wondering what size the band is which appears just above your NICD arrow.

2) How much protein have you loaded per lane? And have you checked for an equal and efficient transfer with Ponceau Red?

3) At which concentration/dilution did you use each lot of the primary antibody? Have you used the same batch of thesecondary antibody?

4) Which film make and detection substrate (ECL, ECL+) have you used?

5) What were the exposuretimes?

6) What have you normalised against (a loading control such as actin or tubulin)? Which software have you used to measure the density/intensity?

7)Have you observedsimilar phenomenons with the previous lots of ab8925?
Once I have received these information, I hope to be able to find an explanation why a new lot of this polyclonal antibody gives slightly lower signal.

I look forward to receiving your reply.